Sudden cardiac death remains a major cause of mortality around the world. Early myocardial ischemia is often a plausible explanation for cardiac arrest in many sudden cardiac death cases but an established postmortem method for its diagnostics is currently lacking. The use of contemporary methods was proposed as a potential diagnostics strategy, specifically evaluation of a panel of molecular tissue markers. Mass spectrometry immunohistochemistry (MS-IHC) and matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI IMS) were investigated as advanced technologies to simultaneously measure multiple molecules in a single tissue section and directly from slide. Both MS-IHC and MALDI IMS successfully detected ischemic areas and allowed not only simultaneous testing of multiple markers but also visualization of molecules' distribution within the tissue by image reconstruction and quantification of the signal. MALDI IMS was also used to characterize molecular changes in response to ischemia by an untargeted approach. Both technologies are powerful mass spectrometry-based tools with a great potential for investigating molecular mechanisms in pathology-related research, accelerating tissue marker discovery, and integrating in routine diagnostics.